Influence of voltage and extracellular Na⁺ on amiloride block and transport kinetics of rat epithelial Na⁺ channel expressed in Xenopus oocytes

Abstract.

We expressed the three subunits of the epithelial amiloride-sensitive Na⁺ channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na⁺ accumulation we performed all experiments in low-Na⁺ solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f _c (2πf _c plot) we determined the rate constants k _on and k _off for the amiloride-ENaC interaction. At a clamp potential (V _m) of –60 mV k _on was 80.8±5.1 µM^–1 s^–1 and k _off 15.4±4.2 s^–1. The half-maximal blocker concentration (K _mic,ami) was 0.19 µM (V _m=–60 mV). While k _on was voltage-independent in the range –50 to –100 mV, k _off and K _mic,ami decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na⁺] ([Na⁺]_o) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na⁺]_o and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na⁺ for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k _on and k _off for amiloride decreased with increasing [Na⁺]_o, while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na⁺]_o, indicating that Na⁺ and amiloride do not compete for the same binding site at the channel.